Peptide conjugates for the stabilization of membrane proteins and interactions with biological membranes

ABSTRACT

The present invention provides a novel class of detergents referred to herein as lipopeptide detergents. Lipopeptide detergents comprise an amphipathic α-helical peptide having a hydrophobic or neutral face and a hydrophilic face. To each end of this peptide is covalently linked an aliphatic hydrocarbon tail, these aliphatic tails being linked thereto such that they associate with the hydrophobic or neutral face of the peptide. Lipopeptide detergents can advantageously be used to stabilize membrane proteins in the absence of a phospholipid bilayer in a manner that preserves the native conformation and permits the subsequent crystallization thereof.

This application claims the benefit of U.S. Provisional Application No.60/140,988, filed Jun. 29, 1999.

FIELD OF THE INVENTION

This invention generally relates to compounds that have utility asdetergents. In particular, the present invention relates to a novelclass of peptide-based chemical compounds that interact with proteins,lipids and other molecules. The compounds may be used for thestabilization and crystallization of proteins and membrane proteins, inparticular. The compounds are also useful for modifying the propertiesof lipid bilayer membranes, and have potential uses as cytolytic agents,as molecules that can facilitate the transport of polar molecules acrossbiological membranes, and as emulsifiers and surfactants.

BACKGROUND OF THE INVENTION

Membrane proteins are critical components of all biological membranes,and can function as enzymes, receptors, channels and pumps. They arealso very common in biological systems, as 20–40% of the genes found inthe bacteria, archaea and eukaryotes code for membrane proteins (Wallinand von Heijne, Protein Sci, 7, 1029–38 (1998), Boyd, et al., ProteinSci, 7, 201–5 (1998), Gerstein, Proteins, 33, 518–34 (1998), Jones, FEBSLett, 423, 281–5 (1998), Arkin, et al., Proteins, 28, 465–6 (1997)).Many clinically useful drugs, including the widely prescribed drugs,fluoxetine (Prozac™) and omeprazole (Prilosec™), interact with humanmembrane proteins. However, despite the abundance and importance ofmembrane proteins, this class of molecules is still only poorlyunderstood at a structural level, mainly because of difficulties ingrowing crystals of membrane proteins suitable for analysis by x-raycrystallography (Garavito, et al., J Bioenerg Biomembr, 28, 13–27(1996), Ostermeier and Michel, Curr Opin Struct Biol, 7, 697–701 (1997),Garavito, Curr Opin Biotechnol 9, 344–349 (1998)).

In order to understand the mechanism of action of a particular membraneprotein, it is essential to know the three-dimensional structure of themolecule to a resolution that reveals its atomic structure. This istypically taken to be better than 0.3 nm resolution, and nearly all ofthe membrane protein structures that are known to this resolution havebeen determined by the technique of x-ray crystallography (Branden andTooze, Introduction to Protein Structure, Garland Publishing Inc., NewYork (1998)). If the protein in question is medically important,knowledge of the 3-dimensional structure of the protein is aprerequisite for the development of new therapeutics usingstructure-based rational drug design methodologies (for example, seeKlabunde, et al., Nature Structural Biology 7, 312–321 (2000)). Thetechniques used in the study of membrane protein crystals are verysimilar to those used for crystals of soluble proteins, and the mainbarrier to advancement in this field is the generation ofdiffraction-quality crystals.

The techniques used for the crystallization of membrane proteins aregenerally similar to the techniques used for the crystallization ofsoluble proteins, and include vapour diffusion, microdialysis and batchmethods (A. McPherson, in “Crystallization of BiologicalMacromolecules”, Cold Spring Harbour Press (1998)). Typically, apurified, concentrated solution of protein is brought to the limit ofits solubility over the course of days or weeks, resulting in either theformation of a protein precipitate or of protein crystals. Becauseprecipitates are more often observed than crystals, numerous conditionsare tested in these trials. The number of trials can vary in number froma few dozen to several thousand in attempts to find conditions resultingin crystal formation. The tested conditions can differ in pH, nature ofadded salts, concentration of the added salts, nature of theprecipitant, concentration of the precipitant, temperature, and otherfactors (A. McPherson, in “Crystallization of BiologicalMacromolecules”, Cold Spring Harbour Press (1998)). In some instances,conditions producing suitable crystals for analysis by x-ray diffractionare not discovered even after extensive screening.

If the protein under consideration is an intrinsic membrane protein, theprotein sample used in the crystallization trials is first purified andstabilized in a specific detergent in order to preserve the nativeconformation of the protein in the absence of a lipid bilayer (H.Michel, Trends Biochem. Sci. 8, 56–59 (1983), W. Kuhlbrandt, Quart. Rev.Biophysics 21, 429–477 (1988)). In most instances, a number of differentdetergents are tested for their ability to stabilize a particularmembrane protein, and for their effect in the crystallization trials.Examples of detergents suitable for these purposes include the alkylgylcoside detergents such as octyl β-D-glucopyranoside (OG, octylglucoside) and dodecyl β-D-maltopyranoside (DDM, dodecyl maltoside)(Baron and Thompson, Biochim. Biophys. Acta 382, 276–285 (1975),Rosevear et al., Biochemistry 19, 4108–4115 (1980)), the polyoxyethylenealkyl ether detergents such as pentaethylene glycol monooctyl ether(C8E5) and octoethylene glycol monododecyl ether (C12E8) (Garavito andRosenbusch, Meth. Enzymol. 125, 309–328 (1886), Victoria and Mahan,Biochim Biophys Acta 644, 226–232 (1981)), and the detergents describedin U.S. Pat. No. 5,674,987, which are prepared from the reaction of acycloalkyl aliphatic alcohol and a saccharide. Detergent-solubilizedmembrane proteins exist as protein-detergent complexes (PDC) in which acluster of detergent molecules covers the surface of the protein that isnormally exposed to the lipophilic core of the lipid bilayer. Thehydrophobic portions of the detergent amphiphiles interact with theprotein surfaces normally in contact with the lipid acyl chains, andthus mimic the normal lipid environment at the surface of the membraneprotein. This micelle-like ring of detergent molecules surrounding themembrane protein is very dynamic and mobile, such that the surfaceproperties of the PDC is in general poorly suited to the formation ofwell-ordered crystals (Crystallization of Membrane Proteins, H. Micheled. CRC Press, Boca Raton, Fla. (1991)). This unfavorable effect islessened in cases where the protein has large extramembranous domains,or with detergents that have small micellar volumes.

A number of techniques have been developed to address this difficulty inattempts to achieve membrane protein crystallization. For example, theformation of a complex with an antibody fragment has been used toincrease the polar surface area of the Paracoccus denitrificanscytochrome oxidase, resulting in well-diffracting crystals (Ostermeieret al., Nat Struct Biol, 2, 842–6 (1995), Ostermeier et al., Proc NatlAcad Sci USA, 94, 10547–53 (1997)). Fusion proteins of the membraneprotein lactose permease with soluble carrier domains have been made inattempts to achieve a similar result (Privé et al., Acta Cryst D50,375–379 (1994), Privé and Kaback, J Bioenerg Biomembr 28, 29–34 (1996)).Bacteriorhodopsin (BR) has been crystallized from cubic lipid phases(Landau and Rosenbusch, Proc Natl Acad Sci USA, 93, 14532–5 (1996)) in amethod that does not rely on detergents at all. However, few crystalssuitable for structure determination have been produced by this method(Chiu, et al., Acta Crystallogr D56, 781–784 (2000)). A strategy toreduce the volume and dynamics of the detergent surface of the PDC hasbeen proposed by Schafineister et al. (Science, 262, 734–8 (1993)). Inthis approach, amphipathic peptides have been used in the place oftraditional detergents such as octyl glucoside. The peptides weredesigned such that the peptide would form an α-helix with onehydrophilic face and one hydrophobic face. The intention was that thehydrophobic surface was of the peptide would associate with thetransmembrane surface of a membrane protein. Although the peptide usedin this study could maintain some membrane proteins in a solubilizedstate for a few days, the proteins were not sufficiently stabilized forthe purposes of crystallization. Because of their limited effectivenessas detergents, these peptides have not found general utility as toolsfor the study of membane proteins.

In the traditional detergents consisting of a polar head group and alinear alkyl tail, the length of the hydrocarbon moiety is an importantfactor in determining the ability of the detergent to preserve thenative conformation of a solubilized membrane protein. Within theframework of a common head group, longer chain length detergents aregenerally more stabilizing towards membrane proteins, and are consideredto be more “gentle”. The presumed mechanism for stabilization is thatthe longer chains are deemed to be more effective at masking thehydrophobic transmembrane surface of the membrane protein than the shortchain detergents and are thus better mimics of the native membraneenvironment. However, longer chain detergents occupy a larger volume ofthe belt region of the PDC, a feature that is expected to reduce theprobability of crystallization of the complex (Michel, 73–87 in“Crystallization of Membrane Proteins”, H. Michel, ed., CRC Press. BocaRaton, Fla. (1991)). Another factor affecting the choice of a particulardetergent is the solubility of the detergent in water or buffersolutions. As the alkyl chain length increases in a series of detergentswith a common head group, the overall solubility of the detergentdecreases, eventually to levels making the detergent impractical formost uses. Thus, octyl glucoside is soluble to levels greater than 20%(w/v) in water, while decyl glucoside is soluble to only 0.1% (w/v) insimilar conditions, and dodecyl glucoside is soluble only to 0.008%(w/v) (Anatrace Inc., Maumee Ohio 1999–2000 Catalogue). With a largerhead group such as maltoside, the solubility of the long chaindetergents increases, but solubility is still reduced to impracticallevels with hexadecyl chain lengths or longer. Thus, within a series oftraditional detergents, there is confict in the preferred length of thealkyl chain length. Long chains favor protein stability, and shortchains are optimal for crystallization and detergent solubility. Sinceprotein stability is a prime concern for crystallization trials, manymembrane protein crystallization trials are carried out undersub-optimal conditions.

Thus, a major use of non-denaturing detergents is for the preservationof the biological function of a membrane protein in the absence of alipid bilayer. These conditions are often encountered during thehandling of membrane proteins, and in particular during the purificationof membrane proteins, and during crystallization trials.

There is a need, thus, for a non-denaturing detergent which effectivelymimics the membrane's lipid bilayer, is capable of solubilizing membraneproteins in such a way that the three-dimensional conformation isretained, and has features to enhance the probablility ofcrystallization of membrane proteins.

SUMMARY OF THE INVENTION

Accordingly, in one aspect, the present invention provides anamphipathic peptide conjugate having detergent properties and having ahydrophobic face and a hydrophilic face, said peptide moiety of theconjugate comprising a first end and a second end, wherein said firstend is covalently linked to a first aliphatic hydrocarbon moiety andsaid second end is covalently linked to a second aliphatic hydrocarbonmoiety, said aliphatic moieties being linked such that they associatewith the peptide moiety of the conjugate.

Preferably the peptide conjugate is a lipopeptide detergent.

Generally, a purified protein in a known detergent is subjected to aprocess whereby the known detergent is exchanged for the novel detergentof the present invention. The protein in the novel detergent may then besubjected to conditions that promote crystallization to occur.

BRIEF DESCRIPTION OF THE DRAWINGS

The present invention is described in further detail herein by referenceto the following drawings in which:

FIG. 1A is a schematic representation of a single lipopeptide detergent(LPD) molecule in accordance with the present invention;

FIG. 1B is a schematic representation of a cylindrical assembly ofseveral lipopeptides in which the aliphatic hydrocarbon tails areclustered in the core of the assembly;

FIG. 1C is a schematic representation of a membrane protein solubilizedby a traditional detergent (prior art);

FIG. 1D is a schematic representation of a membrane protein solubilizedby a lipopeptide detergent in accordance with the present invention;

FIG. 2A is a graph of a series of absorption spectra of the membraneprotein bacteriorhodopsin in the traditional detergent octyl glucoside(OG) showing the loss of the native conformation of the protein overtime;

FIG. 2B is a graph of a series of absorption spectra of the membraneprotein bacteriorhodopsin in the lipopeptide detergent LPD-16 showingthe preservation of the native conformation of the protein over thecourse of 32 days;

FIG. 2C is a graph showing the effectiveness of different concentrationsof the lipopeptide detergent LPD-16 in maintaining the membrane proteinbacteriorhodopsin in a soluble, stable state in the absence of aphospholipid membrane;

FIG. 2D is a graph showing the effectiveness of 5 lipopeptide detergents(LPD-12, LPD-14, LPD-16, LPD-18, LPD-20) in maintaining the membraneprotein bacteriorhodopsin in a soluble, stable state in the absence of aphospholipid membrane; and

FIG. 3 is a histogram demonstrating that the lipopeptide detergentsLPD-12, LPD-14 and LPD-16 interact with phospholipid membranes,dissolving them into micelles.

DETAILED DESCRIPTION OF THE INVENTION

Detailed Description of the Drawings

FIG. 1 is a schematic representation of the lipopeptide detergents. FIG.1A shows a single LPD molecule with the α-helical peptide represented ina Ca tracing with grey lines, and the aliphatic acyl chains of two fattyacids coupled to side chains at either end of the peptide shown withblack lines. This representation is the presumed conformation of themonomer within the assembly shown in FIG. 1B. FIG. 1B shows the presumedassembly of the peptides into a cylindrical assembly. The fatty acylchains cluster in the core of the assembly, near the central axis of thecylinder. FIG. 1C shows a schematic representation of a membrane proteinsolubilized by a traditional detergent. This is included to show thecontrast between the present invention and the prior art. FIG. 1D showsa similar protein solubilized by a lipopeptide detergent.

FIGS. 2 and 3 are discussed in detail later in the description.

The present invention provides novel lipopeptide detergents comprisingan α-helical peptide scaffold having aliphatic hydrocarbon tailscovalently linked to opposite ends of the peptide scaffold.

The peptide scaffold is not particularly limited with respect to itsamino acid sequence.

However, the amino acid sequence is selected so as to permit formationof the peptide scaffold into an amphipathic α-helical conformation.Generally, the peptide will comprise a mixture of hydrophobic andhydrophilic regions. Hydrophobic regions will include, but are notlimited to, neutral or hydrophobic amino acids such as alanine, valine,leucine, isoleucine, methionine, phenylalanine, tryptophane or aminoacids that do not occur in nature. Preferably, the hydrophobic regionsare alanine-rich to favor the formation of an α-helical conformation(Chakrabartty et al., Protein Sci, 3, 843–52 (1994). The hydrophilicregions will include, but are not limited to, amino acids, which areprimarily hydrophilic in nature such as glutamate, lysine, glutamine,aspartate, asparaginine, histidine, serine, tyrosine, threonine or aminoacids that do not occur in nature. Preferably, the hydrophilic regionspromote helix formation through the formation of (i,i+4) salt bridges(Marqusee and Baldwin, Proc Natl Acad Sci USA, 84, 8898–902 (1987)). Thehydrophilic regions of the peptide align on the face of the helix thatwill interact with bulk aqueous phase when in a lipopeptide assembly asshown in FIG. 1B. The neutral or hydrophobic face will include tworesidues for covalent coupling of the aliphatic moieties in the peptideconjugate. These residues will be near the termini of the peptide, atpositions where they are aligned with the hydrophobic face of thepeptide. The two residues can be lysine, ornithine, cysteine, glutamateor aspartate residues, but are not limited to these amino acids.Preferably, the two residues are ornithines.

The number of amino acids in the peptide scaffold is variable, and willgenerally be selected such that the length of the peptide scaffold whenin an α-helical conformation will approximate the width of a naturalmembrane phospholipid bilayer, i.e. between 3.0–4.5 nm. Accordingly, thenumber of amino acids in the peptide scaffold will range from about 15to 35 amino acids. Preferably, the number of amino acids in the scaffoldwill be about 20–30. More preferably, the peptide scaffold will includeabout 25 amino acids, or a number of amino acids which when in anα-helical conformation measure a length of about 3.7 nm.

The terminal amino acids of the peptide scaffold are also selected topromote α-helix formation, and may be naturally occurring amino acids ormodified forms thereof.

Modifications commonly made to terminal amino acids in peptides includethe addition of groups conventionally used in the art of peptidechemistry, which will not adversely affect the function of thelipopeptide. For example, suitable N-terminal blocking groups can beintroduced by alkylation or acylation of the N-terminus. Examples ofsuitable N-terminal blocking groups include C₁–C₅ branched or unbranchedalkyl groups, acyl groups such as formyl and acetyl groups, as well assubstituted forms thereof, such as the acetamidomethyl (Acm) group.Desamino analogs of amino acids are also useful N-terminal blockinggroups, and can either be coupled to the N-terminus of the peptide orused in place of the N-terminal residue. Suitable C-terminal blockinggroups, in which the carboxyl group of the C-terminus is eitherincorporated or not, include esters, ketones or amides. Ester orketone-forming alkyl groups, particularly lower alkyl groups such asmethyl, ethyl and propyl, and amide-forming amino groups such as primaryamines (—NH₂), and mono- and di-alkylamino groups such as methylamino,ethylamino, dimethylamino, diethylamino, methylethylamino and the likeare examples of C-terminal blocking groups. Descarboxylated amino acidanalogues such as agmatine are also useful C-terminal blocking groupsand can be either coupled to the peptide's C-terminal residue or used inplace of it. Further, it will be appreciated that the free amino andcarboxyl groups at the termini can be removed altogether from thepeptide to yield desamino and descarboxylated forms thereof withoutaffect on peptide function. Preferred examples of such modificationsinclude N-terminal acetylation and C-terminal amidation which are knownto promote α-helix formation (Doig et al., Biochemistry, 33, 3396–403(1994)).

Internal amino acids of the peptide may also be modified byderivatization provided that this modification does not affect thefunction of the lipopeptide, and does not interfere with its α-helicalconformation. Such derivatizations can be made to the side chains of theamino acids. For example, the side chains can derivatized byincorporation of blocking groups as described above.

The peptide conjugate may be readily prepared by standard,well-established solid-phase peptide synthesis (SPPS) as described byStewart et al. in Solid Phase Peptide Synthesis, 2nd Edition, 1984,Pierce Chemical Company, Rockford, Ill.; and as described by Bodanszkyand Bodanszky in The Practice of Peptide Synthesis, 1984,Springer-Verlag, New York, and as described in Novabiochem Catalogue andPeptide synthesis handbook, 1997–1998. Other synthetic protocols,including biological or solution phase methods, can also be used. Forthe SPPS method, a suitably protected amino acid residue is firstattached through its carboxyl group to a derivatized, insolublepolymeric support, such as cross-linked polystyrene or polyamide resin.“Suitably protected” refers to the presence of protecting groups on boththe α-amino group of the amino acid, and on any side chain functionalgroups. Side chain protecting groups are generally stable to thesolvents, reagents and reaction conditions used throughout thesynthesis, and are removable under conditions, which will not affect thefinal peptide product. Stepwise synthesis of the oligopeptide is carriedout by the removal of the N-protecting group from the initial aminoacid, and coupling thereto of the carboxyl end of the next amino acid inthe sequence of the desired peptide. This amino acid is also suitablyprotected. The carboxyl of the incoming amino acid can be activated toreact with the N-terminus of the support-bound amino acid by formationinto a reactive group such as formation into a carbodiimide, a symmetricacid anhydride or an “active ester” group such as hydroxybenzotriazoleor pentafluorophenylesters.

Examples of solid phase peptide synthesis methods include the Boc methodwhich utilizes tert-butyloxycarbonyl as the α-amino protecting group,and the Fmoc method which utilizes 9-fluorenylmethyloxycarbonyl toprotect the α-amino of the amino acid residues, both methods of whichare well-known by those of skill in the art.

The aliphatic moieties can be coupled to the resin-coupled peptide byselectively deblocking amino acid side chain protecting groups, followedby reaction with an appropriate aliphatic derivative. Aliphaticderivatives suitable for this purpose include, but are not limited to,saturated fatty acids, unsaturated fatty acids, branched fatty acids,cyclic alkyl acids, aromatic alkyl acids, alkyl amines, alkylmaleimides, alkyl acid chlorides, and alkyl anhydrides. Severalstrategies can be used to couple the aliphatic derivative to thepeptide. For example, if the peptide is synthesized with the Fmocmethod, a Boc group can be used as the protecting group on the δ-aminogroup of the ornithine monomers identified as sites for aliphaticcoupling. Upon completion of the synthesis of the main peptide chain,the ornithine Boc groups can be selectively removed with trifluoroaceticacid, generating free primary amino functionalities at these positions.Reaction with an aliphatic derivative such as a fatty acid can be usedto form an amide linkage with each of the two ornithine side chains.Examples of suitable saturated fatty acids include octanoic acid,nonanoic acid, decanoic acid, undecanoic acid, dodecanoic acid,tridecanoic acid, tetradecanoic acid, pentadecanoic acid, hexadecanoicacid, heptadecanoic acid, octadecanoic acid, nondecanoic acid,eicosanoic acid, heneicosaoic acid, docosanoic acid, tricosanoic acid,tetracosanoic acid, pentacosanoic acid, hexacosanoic acid, heptacosanoicacid and octacosanoic acid. Following the coupling of the aliphaticgroups, the remaining amino acid side chains can be deblocked underappropriate conditions, such as with hydrofluoric acid (HF) ortrifluoromethanesulfonic acid (TFMSA).

Incorporation of N- and/or C-blocking groups can also be achieved usingprotocols conventional to solid phase peptide synthesis methods. Forincorporation of C-terminal blocking groups, for example, synthesis ofthe desired peptide is typically performed using, as solid phase, asupporting resin that has been chemically modified so that cleavage fromthe resin results in a peptide having the desired C-terminal blockinggroup. To provide peptides in which the C-terminus bears a primary aminoblocking group, for instance, synthesis is performed using ap-methylbenzhydrylamine (MBHA) resin so that, when peptide synthesis iscompleted, treatment with HF or TFMSA releases the desired C-terminallyamidated peptide. Similarly, incorporation of an N-methylamine blockinggroup at the C-terminus is achieved using N-methylaminoethyl-derivatizeddivinyl benzene (DVB) resin, which upon treatment with HF releases apeptide bearing an N-methylamidated C-terminus. Blockage of theC-terminus by esterification can also be achieved using conventionalprocedures. This entails use of resin/blocking group combination thatpermits release of side-chain protected peptide from the resin, to allowfor subsequent reaction with the desired alcohol, to form the esterfunction. Fmoc protecting groups, in combination with DVB resinderivatized with methoxyalkoxybenzyl alcohol or equivalent linker, canbe used for this purpose, with cleavage from the support being effectedby trifluoroacetic acid (TFA) in dicholoromethane (DCM). Esterificationof the suitably activated carboxyl function e.g. withN-N′-dicyclohexylcarbodiamide (DCC), can then proceed by addition of thedesired alcohol, followed by deprotection and isolation of theesterified peptide product.

Incorporation of N-terminal blocking groups can be achieved while thesynthesized peptide is still attached to the resin, for instance bytreatment with a suitable anhydride. To incorporate an acetyl blockinggroup at the N-terminus, for instance, the resin-coupled peptide can betreated with 20% acetic anhydride in acetonitrile. The N-blocked peptideproduct can then be cleaved from the resin, deprotected and subsequentlyisolated.

To ensure that the peptide obtained from either chemical or biologicalsynthetic techniques is the desired peptide, analysis of the peptidecomposition should be conducted. Such amino acid composition analysismay be conducted using high resolution mass spectrometry (MS) todetermine the molecular weight of the peptide. Alternatively, oradditionally, the amino acid content of the peptide can be confirmed byhydrolyzing the peptide in aqueous acid, and separating, identifying andquantifying the components of the mixture using reversed-phasehigh-pressure liquid chromatography (HPLC), or an amino acid analyzer.Protein sequenators, which sequentially degrade the peptide and identifythe amino acids in order, may also be used to determine definitely thesequence of the peptide.

Having obtained the desired peptide conjugate, purification to removecontaminants is generally then conducted. Any one of a number ofconventional purification procedures may be used to attain the requiredlevel of purity including, for example, ion-exchange and gel filtrationchromatography or reversed-phase high-pressure liquid chromatography(HPLC) using an alkylated silica column such as C₄-, C₈- or C₁₈-silica.A gradient mobile phase of increasing organic content is generally usedto achieve purification, for example, acetonitrile in an aqueous buffer,usually containing a small amount of trifluoroacetic acid orhydrochloric acid. Because the overall hydrophobicity of the peptideconjugates increases with larger aliphatic moieties, C₄-silica is thepreferred chromatographic resin for these compounds.

Aliphatic hydrocarbon moieties are linked in a covalent manner to boththe N- and C-termini of the scaffold peptide or to sites near each ofthese termini such that they associate with the hydrophobic region ofthe peptide scaffold. In one embodiment of the present invention, thealiphatic hydrocarbon tails are linked to ornithine residues locatedadjacent to N- and C-terminal alanine residues of the scaffold peptide.The δ-amino groups of the ornithines are coupled to the carboxyl groupsof hexadecanoic acid via amide linkages. Ornithines are used in place ofthe more common lysine residues as sites for the hydrocarbon taillinkage since they have fewer methylene groups between the main chainpeptide atoms and the side chain amine, and may position the hydrocarbonchains more precisely in association with the hydrophobic region of thepeptide. Other types of covalent linkages between the peptide scaffoldand the aliphatic hydrocarbon moiety are possible, and can include, butare not limited to, disulfide or ester linkages.

The lipopeptide detergent is advantageous over “traditional” detergentssuch as OG due to its presumed ability to self-associate into a cylinderof defined dimensions. The cylinders are made up of colinear α-helicesand themselves associate into a cylindrical assembly, as shown in FIG.1B, in which the hydrophilic surfaces of the individual helices areexposed to the bulk aqueous phase and the hydrocarbon tails are packedin the core of the assembly effectively mimicking the chains in amembrane phospholipid bilayer. FIG. 1D illustrates how a membraneprotein can be accommodated in the core of a lipopeptide assembly withthe aliphatic hydrocarbon tails forming a cylindrical layer against theprotein, again better mimicking biological membrane conformation,allowing for preservation of the biological activity of solubilizedmembrane proteins.

The described lipopeptide detergents with two coupled aliphatic moietiesranging from ten to twentyfour carbon alkyl chains are soluble in water,in contrast to an alkyl chain length maximum of sixteen carbon groups inthe traditional detergents. The favorable solubility properties of thelong chain lipopeptide detergents make it possible for these detergentsto stabilize large hydrophobic surfaces of membrane proteins.

In addition to their stabilizing properties, the present lipopeptidedetergents have been designed to favor the crystallization of membraneproteins. They lie close to the surface of the membrane protein, and arethus less obtrusive to the formation of a crystal lattice. Also, theypresent a rigid outer surface of α-helices. These are features thatfavor membrane protein crystallization (Schafineister et al., Science,262. 734–8 (1993) Michel, in Crystallization of membrane proteins, 73–87(1991)).

The lipopeptide detergents of the present invention may be used tocrystallize membrane proteins. Generally, the method comprisessolubilizing the membrane protein with a detergent, and then exposingthe solubilized membrane to conditions which promote crystallization tooccur.

The lipopeptide detergents are also membrane-active compounds, and caninsert into phospholipid bilayers. At sufficiently high concentrations,they can disrupt the bilayers and form mixed lipid/lipopeptide micelles.

The lipopeptide detergents of the present invention have the activitiesof traditional detergents and hence they may be used to modulate anddisrupt biological membranes, and therefore to transport polar moleculesacross membranes, including ions. As surface active agents oremulsifiers, they may be used in protein and/or lipid emulsions. Theymay also be used as cytolytic agents.

EXAMPLES

Embodiments of the present invention are described in further detail byreference to the following specific examples, which are not to beconstrued as limiting the appended claims.

Example 1 Synthesis of LPD-16

The lipopeptide, LPD-16, exemplifies a lipopeptide detergent inaccordance with the present invention. The scaffold peptide of LPD-16has the following chemical structure:CH3CONH-AOAEAAEKAAKYAAEAAEKAAKAOA-CONH2wherein A is alanine, 0 is ornithine, E is glutamate, K is lysine, and Yis tyrosine, CH3CONH- is the acetylated amino terminal group of thepeptide, and -CONH2 is the carboxamide end of the peptide chain. Asingle tyrosine is included to allow spectrophotometric detection of thepeptide at 280 nm.

LPD-16 is synthesized on a solid support resin using a combination ofBoc and Fmoc chemistries. The synthesis proceeds from the C-terminus ofthe peptide to the N-terminus, with all the main chain peptide synthesiscouplings based on Fmoc chemistry. The resintert-butoxycarbonyl-Alanine-methylbenzhydrylamine (Boc-Ala-MBHA) ischosen so as to produce a peptide carboxamide upon cleavage from theresin. The resin is first prepared by removal of the Boc protectinggroup with 50% trifluoroacetic acid (TFA), generating the free α-aminoacid amine of the alanine. Sequential addition of the following 24 aminoacids proceeds with the coupling of the appropriate Fmoc-protected aminoacid: N-α-Fmoc-L-alanine (Fmoc-Ala), N-α-Fmoc-L-glutamamic acid α-benzylester (Fmoc-Glu(Obz)), N-α-Fmoc-N-α-2-chloro-benzyloxycarbonyl-L-lysine(Fmoc-Lys(2CIZ)), N-α-Fmoc-N-α-tertbutoxycarbonyl-L-ornithine(Fmoc-Orn(Boc)), or N-α-Fmoc-O-2-bromo-benzyloxycarbonyl-L-tyrosine(Fmoc-Tyr(2BrZ)) with the coupling reagentO-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate(HATU). Upon completion of the coupling reaction, the Fmoc protectinggroup is removed with 20% piperidine in preparation for the next aminoacid coupling. Following the addition of the last amino acid and theremoval of the Fmoc group, the amine terminus of the chain is acetylatedwith acetic anhydride. Next, the Boc protecting groups of the ornithineside chains are removed with 50% TFA in preparation for the couplingwith the fatty acid. Two equivalents of hexadecanoic acid are coupled tothe peptide with HATU. The final step involves the cleavage of thepeptide from the resin and the deprotection of the glutamate, lysine,and tyrosine side chains with trifluoromethanesulfonic acid (TFMSA).

The lipopeptide is precipitated in ether, and washed four times inether. The white pellet is dissolved in water, lyophilized, andredissolved in water. The peptide is purified by gel filtrationchromatography in ammonium carbonate buffer, lyophilized, andredissolved in water. The lipopeptide is then purified by reverse-phaseHPLC on a Waters PrepPak DeltaPak Cartridge (WAT038509, C4, 15 μmparticle size, 300 Å, pore size, 25 mm×100 mm) at a flow rate of 20mLs/min on a Perseptive Biosystems BioCAD HPLC workstation. The elutiongradient is as follows: 2 minutes at 10% solution B/90% solution A, 2minutes with a gradient from 10% to 40% buffer B, 40 minutes with agradient from 40% to 80% buffer B, 2 minutes with a gradient from 80% to90% buffer B. Solution A is 20 mM HCl in HPLC-grade water, and solutionB is 20 mM HCl in acetonitrile. Eluted fractions are collected andanalyzed by Matrix-Assisted Laser-Desorption MassSpectrometry-Time-of-Flight mass spectrometry, and fractions containingthe desired product are pooled and lyophilized to give the final,purifed product.

Lipopeptide detergents with pairs of aliphatic hydrocarbon tails oflength 10, 12, 14, 16, 18, 20, 22, 24, and 28 carbons (LPD-10, LPD-12,LPD-14, LPD-16, LPD-18, LPD-20, LPD-22, LPD-24, and LPD-28) based on thepeptide scaffold CH3CONH-AOAEAAEKAAKYAAEMEKAAKAOA-CONH2 have beendesigned and synthesized by coupling the peptide scaffold to decanoicacid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid,octadecanoic acid, eicosanoic acid, docosanoic acid, tetracosanoic acid,and octacosanoic acid respectively. The LPDs with chain lengths from 10to 16 carbons are soluble in water to over 10 mM, and the LPDs withchain lengths from 18 to 24 carbons are soluble to over 1 mM. LPD-28 ispoorly soluble in water. Computer-assisted molecular modelling suggeststhat the alkyl chains longer than 16 carbons can cross past each otherin LPD-16 through LPD24. As a control, a reference molecule known as C-0is made with the same peptide scaffold, but without the coupled lipids.The C-0 peptide does not have detergent properties. Every batch ofpeptide is analyzed by MS to confirm the synthesis.

Example 2 Effectiveness of Lipopeptides in Stabililizing SolubilizedMembrane Proteins

The membrane protein bacteriorhodopsin was purifed from Halobacteriumsalinarium (gift of J. Lanyi, Univerity of California, Riverside) asfollows. The bacteria were grown in 5 ml Standard Growth Medium (4.28 Msodium chloride, 81.1 mM magnesium sulfate heptahydrate, 10.2 mM sodiumcitrate, 26.8 mM potassium chloride, 10 g/L bacteriological peptone(Oxoid), 1.36 μM calcium chloride, 27.5 μM zinc sulfate heptahydrate, 12μM manganese sulfate, 12 μM ferrous ammonium sulfate hexahydrate, 3.36μM cupric sulfate pentahydrate, pH 7.0) with 1 mg/mL novobiocin withshaking for 5 days at 40° C. 3 mL of this culture were used toinnoculate 300 mL standard growth medium with 1 mg/mL novobiocin, andthe culture was grown for another three days at 40° C. with shaking. 16mL of this culture was used to inoculate 800 mL of Standard Growth Mediawithout novobiocin, and grown for 10 days at 40° C. with shaking. Thecells were harvested by centrifugation at 16000×g for 10 minutes, andthen resuspend in 100 mL 4 M NaCl, 0.5 mg/L DNaseI. The solution wasthen dialyzed with 12–14 kDa molecular weight cutoff (MWCO) membrane(Spectrum Laboratories Inc.) overnight at 4° C. against 12 L 0.1 M NaCl.Membranes were collected by centrifugation of the dialyzed solution at100,000×g for 60 minutes. The membranes were washed 3 times in 0.1 MNaCl by repeatedly homogenizing the membrane pellet in 0.1 M NaCl with aTeflon pestle and centrifuging at 100,000×g for 60 minutes. Purplemembranes were isolated by overlaying 12.5 mL of the membrane suspensionon a 40%/60% (10 mL/7.5 mL) sucrose gradient and centrifuging at75,000×g overnight at 4° C. The purple membranes were removed from thesucrose density gradient and stored at −80° C.

The purple membranes were thawed and diluted 1:20 (v:v) in 0.1 M NaCland spun at 100,000×g for 60 minutes at 4° C. The purple membranes werehomogenized in 25 mM sodium phosphate, pH 6.9 with 1.5% OG (Anatrace,Maumee, Ohio) and mixed for 36 hours in the dark. After adjusting the pHto 5.5 with 0.1 N HCl, the solution was spun at 200,000×g for 45 minutesto obtain the soluble bacterioprhodopsin (BR) in the supernatant. Theprotein was concentrated to 5 mg/mL by ultrafiltration with an Amicon PM10 membrane. BR was further purified from this solution bychromatography on a Superdex 75 gel filtration column in 25 mM sodiumphosphate, pH 5.5, 1.2% (w/v) OG, at a flow rate of 1 mL/min.

OG is a standard detergent for the purification and crystallization ofbacteriorhodopsin (G. F. Schertler et al., J. Mol. Biol 234, 156–164(1993); Landau and Rosenbusch, Proc Natl Acad Sci USA, 93, 14532–5(1996). To exchange the OG for a lipopeptide detergent, the appropriateLPD was added to 300 μL of a 0.5 mg/mL solution of BR in 25 mM NaPO4,1.2% OG, pH 5.5 in a 5000 MWCO Biomax ultrafiltration concentrator. Thesolution was centrifuged at 10,000×g for 5 minutes, reducing the volumeof the retentate to approximately 50 μl. The retentate was then dilutedby the addition of approximately 250 μl of 50 mM NaPO4, 150 mM NaCl, pH7.4, and the concentration/dilution cycle was repeated five times. Theconcentration of OG in the sample was monitored by a colormetric assayfor carbohydrates as described by Dubois et al., Anal. Chem. 28. 350–356(1956). Each concentration/dilution cycle reduced the OG concentrationin the retentate by approximately 65%. Typically, the initialconcentration of OG in the purified BR sample was approximately 50 mM,which is roughly twice the critical micelle concentration for thisdetergent. After three cycles, the concentration of OG was reduced toless than 5 mM, which is near the limit of sensitivity of the OG assay.After five concentration/dilution cycles, there was no detectable OG inthe retentate by the OG assay. The estimated concentration of OG in thefinal sample was approximately 0.5 mM, or approximately 50 times lessthan the critical micelle concentration of OG. The recovery of BR andthe LPD in the retentate was over 90% after five cycles.

The detergent exchanged samples were stored in the dark at roomtemperature, and at 1 day, 4 days, 7 days, 14 days 21 and 32 daysstorage, the samples were centrifuged at 100,000×g for 45 minutes andthe absorption spectrum of the supernatants were were recorded on aPharmacia Ultraspec 200 spectrophotometer from 200 to 700 nm.Solubilized, properly folded, native bacteriorhodopsin remains in thesupernatant and has an absorbance maximum at 550 nm. A control sample ofBR was treated in the same way, except that OG was included in thedilution buffer for each of the 5 rounds of concentration/dilution. Thespectra from a representative experiment with OG is shown in FIG. 2A,and spectra from a sample with LPD-16 is shown in FIG. 2B. The sample inOG lost the characteristic spectum for native bacteriorhodopsin within afew days, while the sample in LPD-16 remained virtually unchanged after32 days.

FIG. 2C shows the result of a similar experiment in which theconcentration of LPD-16 was varied from 0.25 mM to 2.5 mM in the finalsolution. All concentrations were effective at preserving the BR in anative state.

FIG. 2D shows a similar experiment but with different added lipopeptidedetergents (LPD-12, LPD-14, LPD-16, LPD-18 and LPD-20), all at a finalconcentration of 0.5 mM. All were effective in preserving the BR in anative, soluble state. No protein was recovered in control sampleswithout the additon of LPD prior to the five concentration/dilutionsteps (FIGS. 2C and 2D), confirming that the BR is insoluble in theabsense of added detergent. The C-0 control peptide was not effective atmaintaining BR in solution under these conditions (FIG. 2D), anddemonstrates that the presence of the acyl chains on the peptide isessential for the effectiveness of the lipopeptide detergents.

Example 3 Interaction of Lipopeptide Detergents with PhospholipidMembranes

Phosphatidyl choline (PC) vesicles (liposomes) were prepared byextrusion through 0.1 nm pore membranes (Avestin, Ottawa), at 1 mMconcentration in 10 mM N-[2-hydroxyethyl]piperazine-N′-[4-butanesulfonicacid (HEPES), 200 mM NaCl, pH 7.2, and diluted to 0.1 mM phospholipid inthe same buffer. Dodecyl maltoside (DDM), C-0 peptide, or lipopeptidedetergent in the same buffer were added to the indicated concentrationsand the solutions were stored at room temperature for 24 hours. Thehydrodynamic radius (Rh) and polydispersity of the solutions weremeasured on a DynaPro-800 dynamic light scattering device (ProteinSolutions Inc., Charlottesville, Va.). Estimates on the error on the Rhvalues were taken as the polydispersity, as recommended by themanfacturer. Samples of DDM, C-0 peptide and LPD in the absence of PCliposomes were also analyzed.

The results of this experiment are illustrated in FIG. 3. The histogramdemonstrates that the lipopeptide detergents LPD-12, LPD-14 and LPD-16interact with phospholipid membrane vesicles, dissolving them intomicelles. The control C-0 peptide had no measureable effect on thesevesicles. The control sample with DDM confirms that the traditionaldetergent can also effect a transition. The initial liposomes have an Rhvalue of approximately 3240 nm, and the micelles have an Rh of 2.54.5nm. The final concentration of DDM, peptide or lipopeptide in thesamples were as follows: DDM with PC liposomes, 0.8 mM; DDM withoutliposomes, 1.0 mM; C-0 with PC liposomes, 2.0 mM; C-0 without liposomes,2.2 mM; LPD-12 with PC liposomes, 1.25 mM; LPD-12 without liposomes, 1mM; LPD-14 with PC liposomes, 1.5 mM; LPD-14 without liposomes, 0.9 mM;LPD-16 with PC liposomes, 1 mM; LPD-16 without liposomes, 0.9 mM. Theexact Rh values did not depend strongly on the exact concentration ofthe added DDM or lipopeptide, as long as it was above the criticalthreshold value to effect the transition from liposomes to micelles inthe samples with 0.1 mM PC.

The disclosures of all of the literature references and any patentsreferred to herein are incorporated herein by reference.

While the invention has been described with particular reference tocertain embodiments thereof, it will be understood that those ofordinary skill in the art within the scope and spirit of the followingclaims may make changes and modifications.

In the claims, the word “comprising” means “including the followingelements (in the body), but not excluding others”; the phrase“consisting of” means “excluding more than traces of other than therecited ingredients”; and the phrase “consisting essentially of” means“excluding unspecified ingredients which materially affect the basiccharacteristics of the composition”.

1. A lipopeptide detergent comprising a peptide moiety having the aminoacid sequence AOAEAAEKAAKYAAEAAEKAAKAOA (SEQ ID NO: 1) and comprising afirst end and a second end, wherein said first end is covalently linkedto a first aliphatic hydrocarbon moiety and said second end iscovalently linked to a second aliphatic hydrocarbon moiety, saidaliphatic moieties being linked such that they associate with ahydrophobic region of the peptide moiety.
 2. The lipopeptide detergentas defined in claim 1, wherein said peptide comprises hydrophobic andhydrophilic regions.
 3. The lipopeptide detergent as defined in claim 1,wherein said peptide comprises 15–35 amino acids.
 4. The lipopeptidedetergent as defined in claim 3, wherein said peptide comprises about 25amino acids.
 5. The lipopeptide detergent as defined in claim 1, whereinthe length of said peptide is approximately equal to the width of aphospholipid bilayer.
 6. The lipopeptide detergent as defined in claim5, wherein the length of said peptide is in the range of about 3.5–4.0nm.
 7. The lipopeptide detergent as defined in claim 6, wherein thelength of said peptide is about 3.7 nm.
 8. The lipopeptide detergent asdefined in claim 1, wherein the termini of said peptide are protected.9. The lipopeptide detergent as defined in claim 8, wherein theN-terminus of said peptide is acetylated and the C-terminus of saidpeptide is amidated.
 10. The lipopeptide detergent as defined in claim1, wherein said aliphatic hydrocarbon moieties each comprise from about8–24 carbon atoms.
 11. The lipopeptide detergent as defined in claim 1,wherein said detergent is comprised of a peptide scaffoldCH3CONH-AOAEAAEKAAKYAAEAAEKAAKAOA (SEQ ID NO: 1)-CONH2 coupled at eachend to an aliphatic fatty acid selected from the group consisting ofdecanoic acid, dodecanoic acid, tetradecanoic acid, hexadecanoic acid,octadecanoic acid, eicosanoic acid, docosanoic acid, tetracosanoic acid,and octacosanoic acid.
 12. The lipopeptide detergent as defined in claim1, wherein each of said aliphatic hydrocarbon moieties is an aliphatichydrocarbon tail having a length of from 10 to 28 carbon atoms.
 13. Thelipopeptide detergent as defined in claim 12, wherein each of saidaliphatic hydrocarbon moieties is an aliphatic hydrocarbon tail having alength of 16 carbon atoms.
 14. The lipopeptide detergent as defined inclaim 12, wherein each of said aliphatic hydrocarbon moieties is analiphatic hydrocarbon tail having a length of 12 carbon atoms.
 15. Alipopeptide detergent as defined in claim 1, wherein said aliphatichydrocarbon moieties are covalently linked to the peptide moiety at theornithine residues of the peptide moiety.
 16. A composition comprisingmembrane proteins stabilized by a lipopeptide detergent as defined inclaim
 1. 17. A composition comprising a biological membrane treated witha lipopeptide detergent as defined in claim 1.